Lung Squamous Cell Carcinoma Tumor Research Articles

FP16.01 Development and Characterization of Two Novel Squamous Cell Lung Cancer Mouse Cell Lines

Lung cancer is the leading cause of cancer-related death worldwide. The incidence and mortality associated with lung cancer is a major Public Health challenge in advanced societies. About 20-30% of all lung cancers are classified as lung squamous cell carcinoma (LUSC), also known as squamous cell carcinoma (SSC or SqCC) or epidermoid carcinoma. The absence of preclinical models makes LUSC disease analysis and research more challenging. The development of well-characterized cellular and molecular tools is essential for the advance in lung cancer research. In the present work, we have performed an exhaustive molecular and functional characterization of a new LUSC research model, recently developed in our laboratory. This model consists of two novel A/J mouse-derived syngeneic cell lines from NTCU-induced LUSC.In our opinion, these cell lines may become a robust tool for the study of squamous cell lung cancer in a reliable and reproducible manner. Carcinogenesis and cell line generation: LUSC tumors were induced by N-nitroso-tris-chloroethylurea (NTCU) (Toronto Research Chemicals) treatment applying 0.04 M NTCU by skin painting twice a week for 20 weeks to 8-week-old A/J mice. Immunohistochemical staining: Dissected tumors were fixed in 4% paraformaldehyde and embedded in paraffin. Three-micrometer-thick sections were used for tumors immunohistochemical phenotypic characterization. Exome sequencing: Whole exome sequencing was performed on extracted DNA from the tumor-derived cell lines. RNA sequencing analysis: Samples were prepared with the Illumina TruSeq Stranded mRNA kit and sequenced as reverse paired-end (50 bp) runs on the Nextseq sequencer. Tumor immunotherapy experiments: 2x106 cells were subcutaneously inoculated in one flank of 8-week-old female A/J mice. When the tumors reached a volume of 75 mm3, mice were randomized and treated intraperitoneally with 3 doses of 100 μg of anti-PD1 or anti-CTLA4. Tumor immune landscape analysis: 2x106 cells were subcutaneously inoculated in one flank of 6 8-week-old female A/J mice . Thirteen days after cell inoculation, tumors were collected and processed for flow cytometry analysis. Metastasis in vivo model: Cells were transduced with a triple modality construct containing a triple fusion protein GFP, luciferase and thymidine kinase. Eight-week old female mice were inoculated in the left cardiac ventricle with 1 × 105 cells. Cells were followed by bioluminescence. We have generated two transplantable LUSC cell lines (UN-SCC679 and UN-SCC680) derived from an N-nitroso-tris-chloroethylurea (NTCU) chemically-induced mouse model in A/J mice. Both cells lines expressed cytokeratins and p40, and lacked thyroid transcription factor 1 (TTF1) expression. Furthermore, we genetically characterized the LUSC cell lines by performing whole exome and RNA sequencing. In addition, we characterized the immune landscape of both tumors in vivo and assessed their response to immunotherapy studies combining the gold-standard checkpoint inhibitors in clinic. Finally, we studied the metastatic potential of each of these two models and confirmed that they reflect the human LUSC organotropism to the brain, bones, liver and adrenal glands. We have generated a very valuable tool for LUSC research that recapitulates the complexity of the human disease.

Comprehensive Analysis of the Prognostic Role and Mutational Characteristics of m6A-Related Genes in Lung Squamous Cell Carcinoma.

BackgroundThere have been limited treatment therapies for lung squamous cell carcinoma (LUSC). M6A-related genes may be the next therapeutic targets for LUSC. In this study, we explored the prognostic role and mutational characteristics of m6A-related genes in LUSC.MethodsLUSC gene expression data, mutational data, and corresponding clinical information were extracted from The Cancer Genome Atlas database. Differentially expressed genes (DEGs) were identified, and the mutation characteristics of LUSC patients were explored. Then, m6A-related genes were extracted and the correlations among the genes were detected. Finally, the prognostic roles of the genes were investigated and the nomogram model was developed. Besides, the protein–protein interaction (PPI) network was used to explore the potential interactions among the genes.ResultsIn total, there are 551 LUSC samples enrolled in our study, containing 502 LUSC tumor samples and 49 adjacent normal LUSC samples, respectively. There were 2970 upregulated DEGs and 1806 downregulated DEGs were further explored. IGF2BP1 and RBM15 had significant co-occurrence frequency (p < 0.05). Besides, METTL14 and ZC3H13 or YTHDF3 also had significant co-occurrence frequency (p < 0.05). All the m6A-related genes represent the positive correlation. WTAP was identified as a prognostic gene in the TCGA database while YTHDC1 and YTHDF1 were identified as prognostic genes. In multivariate Cox analysis, YTHDF1, age, pN stage, pTNM stage, and smoking were all identified as significant prognostic factors for OS.ConclusionWe investigated the expression patterns and mutational characteristics of LUSC patients and identified three potential independent prognostic m6A-related genes (WTAP, YTHDC1, and YTHDF1) for OS in LUSC patients.

Molecular characterization of lung squamous cell carcinoma tumors reveals therapeutically relevant alterations.

Introduction: Unlike lung adenocarcinoma patients, there is no FDA-approved targeted-therapy likely to benefit lung squamous cell carcinoma patients.Materials and Methods: We performed survival analyses of lung squamous cell carcinoma patients harboring therapeutically relevant alterations identified by whole exome sequencing and mass spectrometry-based validation across 430 lung squamous tumors.Results: We report a mean of 11.6 mutations/Mb with a characteristic smoking signature along with mutations in TP53 (65%), CDKN2A (20%), NFE2L2 (20%), FAT1 (15%), KMT2C (15%), LRP1B (15%), FGFR1 (14%), PTEN (10%) and PREX2 (5%) among lung squamous cell carcinoma patients of Indian descent. In addition, therapeutically relevant EGFR mutations occur in 5.8% patients, significantly higher than as reported among Caucasians. In overall, our data suggests 13.5% lung squamous patients harboring druggable mutations have lower median overall survival, and 19% patients with a mutation in at least one gene, known to be associated with cancer, result in significantly shorter median overall survival compared to those without mutations. Conclusions: We present the first comprehensive landscape of genetic alterations underlying Indian lung squamous cell carcinoma patients and identify EGFR, PIK3CA, KRAS and FGFR1 as potentially important therapeutic and prognostic target.

Schlafen 12 Is Prognostically Favorable and Reduces C-Myc and Proliferation in Lung Adenocarcinoma but Not in Lung Squamous Cell Carcinoma.

Simple SummaryDifferent subtypes of lung cancer respond differently to treatment. To understand the signaling pathways that dictate the aggressive behavior of lung adenocarcinoma vs. lung squamous cell carcinoma, we performed a survival analysis that demonstrated that the protein Schlafen 12 correlates with better survival in patients with lung adenocarcinoma but not patients with lung squamous cell carcinoma, indicating specificity of the effect of the SLFN12 pathway in lung cancer subtypes. We investigated this specific effect by confirming the ability of Schlafen 12 to reduce cell proliferation of lung adenocarcinoma cells in vitro but not that of lung squamous cell carcinoma. Moreover, we demonstrated that Schlafen 12 acts through translational inhibition of c-myc protein in lung adenocarcinoma cells. The ability to delineate the exact signaling pathways that modulate each lung cancer subtype’s aggressive behavior will help in the future development of precision medicine to target this challenging disease.Schlafen 12 (SLFN12) is an intermediate human Schlafen that induces differentiation in enterocytes, prostate, and breast cancer. We hypothesized that SLFN12 influences lung cancer biology. We investigated survival differences in high versus low SLFN12-expressing tumors in two databases. We then adenovirally overexpressed SLFN12 (AdSLFN12) in HCC827, H23, and H1975 cells to model lung adenocarcinoma (LUAD), and in H2170 and HTB-182 cells representing lung squamous cell carcinoma (LUSC). We analyzed proliferation using a colorimetric assay, mRNA expression by RT-qPCR, and protein by Western blot. To further explore the functional relevance of SLFN12, we correlated SLFN12 with seventeen functional oncogenic gene signatures in human tumors. Low tumoral SLFN12 expression predicted worse survival in LUAD patients, but not in LUSC. AdSLFN12 modulated expression of SCGB1A1, SFTPC, HOPX, CK-5, CDH1, and P63 in a complex fashion in these cells. AdSLFN12 reduced proliferation in all LUAD cell lines, but not in LUSC cells. SLFN12 expression inversely correlated with expression of a myc-associated gene signature in LUAD, but not LUSC tumors. SLFN12 overexpression reduced c-myc protein in LUAD cell lines but not in LUSC, by inhibiting c-myc translation. Our results suggest SLFN12 improves prognosis in LUAD in part via a c-myc-dependent slowing of proliferation.

Abstract 1671: Deregulation of mTOR signaling contributes to chemoradiation resistance in lung squamous cell carcinoma

Abstract Non-small cell lung carcinoma (NSCLC) represents ~ 85% of lung cancer, and ~1/3 of these patients present with locally-advanced, unresectable/medically-inoperable disease. Patients who present with locally-advanced NSCLC are commonly treated with definitive chemoradiation. Even though an initial response to concurrent chemoradiation is commonly observed, disease relapse in the radiation fields occurs in 1/3 of cases, likely driven by therapy-resistant tumor cells. However, the underlying molecular mechanisms of chemoradiation resistance of NSCLC remains largely unknown. We herein report that upregulation of mTOR signaling plays an important role in the chemoradiation resistance of NSCLC. Patient-derived xenograft (PDX) models of lung squamous cell carcinoma (LUSC) were established in the flanks of NOD-SCID gamma (NSG) mice. Chemoradiation resistant tumor models were developed by treating the tumors with a regimen of 2-4 weeks of chemoradiation (20, 30 or 40 Gy at 2 Gy per day, concurrent with cisplatin). Recurrent tumor models were collected and subjected to functional proteomics using reverse phase protein analysis (RPPA). Several LUSC tumors recurred in the 20 Gy+cisplatin, and 40 Gy+cisplatin groups. RPPA analysis of the recurrent and parental (untreated) PDX tumors showed that there were enhanced mTOR signaling in the chemoradiation resistant 20 Gy+cisplatin and 40 Gy+cisplatin tumors compared to that in the control tumors. Of note, phospho-AKT-S473, phospho-AKT-S308, phospho-S6-S235/6 and phospho-GSK3β-S9/21 were the highest upregulated signaling and were validated by immunoblotting. Consistently, pharmacological inhibition of mTOR kinase by Torin2 significantly sensitized LUSC cell lines to ionizing radiation. Our results demonstrate that utilization of PDX models is a useful method to elucidate the molecular mechanisms of chemoradiation resistance of NSCLC and deregulation of mTOR signaling contributes to the chemoradiation resistance of NSCLC. Our findings suggest that clonal selection of subpopulations with high mTOR signaling in heterogeneous NSCLC tumors might contribute to the relapse of NSCLC after chemoradiation and that mTOR kinase inhibitors are promising agents for the upfront treatment of NSCLC patients in combination with chemoradiation. Citation Format: Changxian Shen, Duan-Liang Shyu, Min Xu, Linlin Yang, Wenrui Duan, Terence M. Williams. Deregulation of mTOR signaling contributes to chemoradiation resistance in lung squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1671.

Downregulation of miR-125b-5p and Its Prospective Molecular Mechanism in Lung Squamous Cell Carcinoma.

Background: To explore the clinical significance of miR-125b-5p and its potential mechanisms in lung squamous cell carcinoma (LUSC). Materials and Methods: An integrated analysis of data from in-house quantitative real-time polymerase chain reaction (qRT-PCR), microRNA-sequencing, and microarray assays to appraise the expression level of miR-125b-5p in LUSC tissues compared to adjacent noncancerous controls. The authors identified the candidate targets of miR-125b-5p and conducted functional analysis using computational biology strategies from gene ontology, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, disease ontology (DO), and protein-protein interaction (PPI) network analyses to investigate the prospective mechanisms. Results: According to qRT-PCR results, the expression level of miR-125b-5p was markedly decreased in LUSC tissues compared to noncancerous control tissues. Receiver operating characteristic and summary receiver operating characteristic analyses showed that miR-125b-5p had good specificity and sensitivity for distinguishing LUSC tissue from noncancerous lung tissue. The standard mean difference revealed that men and women with lower expression levels of miR-125b-5p may have a higher risk for LUSC. KEGG analysis and DO analysis intimated that target genes were evidently enriched in pyrimidine metabolism and pancreatic carcinoma. The PPI network of the top assembled KEGG pathway indicated that RRM2, UMPS, UCK2, and CTPS1 were regarded as crucial target genes for miR-125b-5p, and RRM2 was eventually deemed a key target. Conclusions: The authors' findings implicate a low expression level of miR-125b-5p in LUSC. A tumor-suppressive role of miR-125b-5p is proposed, based on its effects on LUSC tumor growth, clinical stage progression, and lymph node metastasis.

Generation of Genetically Engineered Mouse Lung Organoid Models for Squamous Cell Lung Cancers Allows for the Study of Combinatorial Immunotherapy.

Lung squamous cell carcinoma (LSCC) is a deadly disease for which only a subset of patients responds to immune checkpoint blockade (ICB) therapy. Therefore, preclinical mouse models that recapitulate the complex genetic profile found in patients are urgently needed. We used CRISPR genome editing to delete multiple tumor suppressors in lung organoids derived from Cre-dependent SOX2 knock-in mice. We investigated both the therapeutic efficacy and immunologic effects accompanying combination PD-1 blockade and WEE1 inhibition in both mouse models and LSCC patient-derived cell lines. We show that multiplex gene editing of mouse lung organoids using the CRISPR-Cas9 system allows for efficient and rapid means to generate LSCCs that closely mimic the human disease at the genomic and phenotypic level. Using this genetically defined mouse model and three-dimensional tumoroid culture system, we show that WEE1 inhibition induces DNA damage that primes the endogenous type I IFN and antigen presentation system in primary LSCC tumor cells. These events promote cytotoxic T-cell-mediated clearance of tumor cells and reduce the accumulation of tumor-infiltrating neutrophils. Beneficial immunologic features of WEE1 inhibition are further enhanced by the addition of anti-PD-1 therapy. We developed a mouse model system to investigate a novel combinatory approach that illuminates a clinical path hypothesis for combining ICB with DNA damage-inducing therapies in the treatment of LSCC.

An 11-lncRNA risk scoring model predicts prognosis of lung squamous cell carcinoma.

The study aims to construct a multi-gene risk scoring model that can be used to predict the prognosis of patients with lung squamous cell carcinoma (LUSC). RNA-seq data from 494 LUSC tumor samples and 49 peripheral lung tissue samples were obtained from TCGA_LUSC database. Differential analysis was conducted using edgeR to screen differentially expressed lncRNAs (DElncRNAs) between LUSC and normal samples. Univariate Cox regression analysis was used to screen lncRNAs that were significantly correlated with LUSC prognosis. LASSO regression model was built to reduce complexity. The LUSC prognostic model based on lncRNAs was established by multivariate Cox regression analysis, which was evaluated by ROC curves and survival analysis. ROC and Kaplan-Meier survival curves of each lncRNA in the model were plotted and compared. 2085 DElncRNAs were identified. Combined with univariate Cox regression analysis, 342 prognosis-related genes were screened. After LASSO regression analysis, 11 lncRNAs tightly related to LUSC prognosis were identified and a risk scoring model was constructed. ROC curve analysis proved the good performance of the model. The Kaplan-Meier survival curve showed that the mortality in high-risk group was significantly higher. The survival analysis results of each lncRNA were also consistent with the prediction in Cox regression. Our results suggested that the 11-lncRNA risk scoring model may provide a new insight for predicting prognosis of LUSC patients.

A23 A Genomically Adjusted Clinicopathologic Model Predicts Recurrence in Resected Early-Stage Lung Squamous Cell Carcinoma

In contrast to lung adenocarcinoma, identification of clinically relevant genomic perturbations in lung squamous cell carcinoma (LUSC) remains poorly characterized. Prognostic and therapeutic decisions following surgery in early and locoregionally advanced LUSC are almost exclusively driven by the TNM classification system, omitting high-risk clinicopathologic and tumor genomic information. To address this knowledge gap, we sought to determine if a combined clinicopathologic and genomic model could predict disease-free survival (DFS) better than traditional TNM assessments in completely resected LUSC. A retrospective cohort study of a prospectively maintained database was performed for patients (N=95) with pathologic stage I-III LUSC who underwent complete resection from 2008-2018. Patients who received any induction therapy (N=9) were excluded. All patients had complete clinicopathologic data with broad-panel next-generation sequencing of the primary tumor, including matched controls to bioinformatically filter germline variants. DFS, the primary endpoint, and overall survival (OS) were estimated using Kaplan-Meier. Genomic pathway alterations (N=10) were determined as we previously reported. The prognostic model established associations between high-risk clinicopathologic variables and genomic alterations with DFS through Cox regression models. Concordance probability estimate (CPE) was used to discriminate performance between the existing TNM model and the developed prediction models. The median age was 70 years (range, 55-84), one-third were female (n=35), 55% (N=52) were pathologic stage I, and 98% (N=93) were ever-smokers. Median follow-up was 2 years. Recurrence occurred in 20% (19/95) and DFS was 74% (95% CI, 84-96%) at two years. Clinicopathologic features associated with DFS were lymphovascular invasion, visceral-pleural invasion, and pathologic stage. Tumor genomic analysis revealed alterations in the transcription factor BCL6 were independently associated with worse DFS (HR 5.23, 95% CI 1.73, 15.9, p=0.009), while mutations in the tumor suppressor ARID1A were associated with a worse OS (HR 2.98, 95% CI 0.91, 9.77, p=0.07). Pathway-centric analyses revealed no associations with our primary or secondary endpoints. Our prognostic clinicopathologic model outperformed the internally validated TNM model (CPE, 0.74 vs. 0.70) for prediction of DFS and our clinicopathologic model, adjusted for BCL6 genomic alterations, further improved discrimination (CPE=0.77). We show that integration of high-risk clinicopathologic and tumor genomic profiling better predicts DFS than TNM classification alone in early-stage, surgically resected LUSC – an observation that may facilitate enrichment in future adjuvant therapy clinical trials. This exploratory genomic analysis also suggests future studies investigate putative therapeutic vulnerabilities in LUSC tumors harboring BCL6 and ARD1A genomic alterations.

Higher prevalence of homologous recombination deficiency in tumors from African Americans versus European Americans.

To improve our understanding of longstanding disparities in incidence and mortality in lung cancer across ancestry, we performed a systematic comparative analysis of molecular features in tumors from African Americans (AAs) and European Americans (EAs). We find that lung squamous cell carcinoma tumors from AAs exhibit higher genomic instability-the proportion of non-diploid genome-aggressive molecular features such as chromothripsis and higher homologous recombination deficiency (HRD). In The Cancer Genome Atlas, we demonstrate that high genomic instability, HRD and chromothripsis among tumors from AAs is found across many cancer types. The prevalence of germline HRD (that is, the total number of pathogenic variants in homologous recombination genes) is higher in tumors from AAs, suggesting that the somatic differences observed have genetic ancestry origins. We also identify AA-specific copy-number-based arm-, focal- and gene-level recurrent features in lung cancer, including higher frequencies of PTEN deletion and KRAS amplification. These results highlight the importance of including under-represented populations in genomics research.

MiR-223-3p functions as a tumor suppressor in lung squamous cell carcinoma by miR-223-3p-mutant p53 regulatory feedback loop

BackgroundMicroRNAs have an important role in diverse biological processes including tumorigenesis. MiR-223 has been reported to be deregulated in several human cancer types. However, its biological role has not been functionally characterized in lung squamous cell carcinoma (LSCC). The following study investigates the role of miR-223-3p in LSCC growth and metastasis and its underlying mechanism.MethodsMicroRNA profiling analyses were conducted to determine differential miRNAs expression levels in LSCC tumor tissues that successfully formed xenografts in immunocompromised mice (XG) and failed tumor tissues (no-XG). RT-PCR and in situ hybridization (ISH) was performed to evaluate the expression of miR-223-3p in 12 paired adjacent normal tissues and LSCC specimens. Cell proliferation and migration were assessed by CCK-8, colony formation and Transwell assay, respectively. The role of miR-223-3p in LSCC tumorigenesis was examined using xenograft nude models. Bioinformatics analysis, Dual-luciferase reporter assays, Chromatin immunoprecipitation (ChIP) assay and Western blot analysis were used to identify the direct target of miR-223-3p and its interactions.ResultsMiR-223-3p was downregulated in LSCC tissues that successfully formed xenografts (XG) compared with tumor tissues that failed (no-XG), which was also significantly reduced in LSCC tissues compared with the adjacent normal tissues. Gain- and loss-of function experiments showed that miR-223-3p inhibited proliferation and migration in vitro. More importantly, miR-223-3p overexpression greatly suppressed tumor growth in vivo. Mechanistically, we found that mutant p53 bound to the promoter region of miR-223 and reduced its transcription. Meanwhile, p53 is a direct target of miR-223-3p. Thus, miR-223-3p regulated mutant p53 expression in a feedback loop that inhibited cell proliferation and migration.ConclusionsOur study identified miR-223-3p, as a tumor suppressor gene, markedly inhibited cell proliferation and migration via miR-223-3p-mutant p53 feedback loop, which suggested miR-223-3p might be a new therapeutic target in LSCC bearing p53 mutations.

LUBAC determines chemotherapy resistance in squamous cell lung cancer.

Lung squamous cell carcinoma (LSCC) and adenocarcinoma (LADC) are the most common lung cancer subtypes. Molecular targeted treatments have improved LADC patient survival but are largely ineffective in LSCC. The tumor suppressor FBW7 is commonly mutated or down-regulated in human LSCC, and oncogenic KRasG12D activation combined with Fbxw7 inactivation in mice (KF model) caused both LSCC and LADC. Lineage-tracing experiments showed that CC10+, but not basal, cells are the cells of origin of LSCC in KF mice. KF LSCC tumors recapitulated human LSCC resistance to cisplatin-based chemotherapy, and we identified LUBAC-mediated NF-κB signaling as a determinant of chemotherapy resistance in human and mouse. Inhibition of NF-κB activation using TAK1 or LUBAC inhibitors resensitized LSCC tumors to cisplatin, suggesting a future avenue for LSCC patient treatment.

Gemcitabine resistance mediated by ribonucleotide reductase M2 in lung squamous cell carcinoma is reversed by GW8510 through autophagy induction

Although chemotherapeutic regimen containing gemcitabine is the first-line therapy for advanced lung squamous cell carcinoma (LSCC), gemcitabine resistance remains an important clinical problem. Some studies suggest that overexpressions of ribonucleotide reductase (RNR) subunit M2 (RRM2) may be involved in gemcitabine resistance. We used a novel RRM2 inhibitor, GW8510, as a gemcitabine sensitization agent to investigate the therapeutic utility in reversing gemcitabine resistance in LSCC. Results showed that the expressions of RRM2 were increased in gemcitabine intrinsic resistant LSCC cells upon gemcitabine treatment. GW8510 not only suppressed LSCC cell survival, but also sensitized gemcitabine-resistant cells to gemcitabine through autophagy induction mediated by RRM2 down-regulation along with decrease in dNTP levels. The combination of GW8510 and gemcitabine produced a synergistic effect on killing LSCC cells. The synergism of the two agents was impeded by addition of autophagy inhibitors chloroquine (CQ) or bafilomycin A1 (Baf A1), or knockdown of the autophagy gene, Bcl-2-interacting protein 1 (BECN1). Moreover, GW8510-caused LSCC cell sensitization to gemcitabine through autophagy induction was parallel with impairment of DNA double-strand break (DSB) repair and marked increase in cell apoptosis, revealing a cross-talk between autophagy and DNA damage repair, and an interplay between autophagy and apoptosis. Finally, gemcitabine sensitization mediated by autophagy induction through GW8510-caused RRM2 down-regulation was demonstrated in vivo in gemcitabine-resistant LSCC tumor xenograft, further indicating that the sensitization is dependent on autophagy activation. In conclusion, GW8510 can reverse gemcitabine resistance in LSCC cells through RRM2 downregulation-mediated autophagy induction, and GW850 may be a promising therapeutic agent against LSCC as it combined with gemcitabine.

Novel mutational landscapes and expression signatures of lung squamous cell carcinoma.

Lung squamous cell carcinoma (LUSC) is a major subtype of Non-Small Cell Lung Cancer. To increase our understanding of the LUSC pathobiology, we performed exome sequencing and RNA-seq in 16 murine carcinogen-induced LUSC tumors and 8 normal murine lung tissue samples. Additionally, we conducted single-cell RNA-seq on two independent tumors from the same murine model. We identified a list of 59 cancer genes recurrently mutated in the mice LUSC tumors, 47 (80%) of which were also mutated in human LUSCs. At the single cell level, we detected unique clonal mutation patterns for each of the two LUSC tumors, being initiated from clones carrying the mutant Igfbp7 and Trp53 genes, respectively. We also identified an expression signature serving as an effective classifier for LUSC tumors and a strong predictor of survival outcomes of lung cancer patients. Lastly, we found that some of the mutant LUSC genes were associated with the significantly altered tumoral expression of inhibitory immune checkpoint genes such as PD-L1, VISTA, TIM3 and LAG3 in human LUSCs. The novel findings of clonal evolution, mutational landscapes and expression signatures of LUSC suggested new targets for the overall LUSC therapy and the immunotherapy of LUSC.

FGF19 genetic amplification as a potential therapeutic target in lung squamous cell carcinomas.

BackgroundAlthough FGF 19 gene aberrations are associated with carcinogenesis and progression in human cancers, the roles of FGF 19 genetic amplification and expression in Chinese patients with lung squamous cell carcinoma (LSCC) and FGF 19 amplification as a potential therapeutic target for LSCC are not well understood.MethodsFluorescence in situ hybridization analysis and quantitative real‐time‐PCR was used to detect FGF 19 genetic amplification and FGF 19 messenger RNA expression in LSCC tumor and paired adjacent samples. Small interfering RNA and short hairpin RNA were used to knockdown FGF19 in vitro and in vivo. Results FGF 19 amplification was identified in a subset of LSCC patients (37.5%, 15/40), and upregulation of FGF 19 expression was found in 60% (24/40) of tumor tissues compared to adjacent non‐tumorous tissues. Correlation analysis with clinicopathologic parameters showed that FGF 19 upregulation was significantly associated with heavy smoking. Small interfering RNA knockdown of FGF 19 led to the significant inhibition of cell growth and induced apoptosis in LSCC cells carrying the amplified FGF 19 gene, but these effects was not observed in non‐amplified LSCC cells. Interfering FGF 19 expression with short hairpin RNA also resulted in tumor growth inhibition and induced apoptosis in LSCC xenografts with amplified FGF 19 in tumor cells.ConclusionOur results suggested that FGF 19 signaling activation is required for cell growth and survival of FGF 19 amplified LSCC cells, both in vitro and in vivo. Intervention of FGF 19 activation could be a potential therapeutic strategy for LSCC patients with FGF 19 amplification.

Genome-wide copy number variation pattern analysis and a classification signature for non-small cell lung cancer.

The accurate classification of non-small cell lung carcinoma (NSCLC) into lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) is essential for both clinical practice and lung cancer research. Although the standard WHO diagnosis of NSCLC on biopsy material is rapid and economic, more than 13% of NSCLC tumors in the USA are not further classified. The purpose of this study was to analyze the genome-wide pattern differences in copy number variations (CNVs) and to develop a CNV signature as an adjunct test for the routine histopathologic classification of NSCLCs. We investigated the genome-wide CNV differences between these two tumor types using three independent patient datasets. Approximately half of the genes examined exhibited significant differences between LUAD and LUSC tumors and the corresponding non-malignant tissues. A new classifier was developed to identify signature genes out of 20 000 genes. Thirty-three genes were identified as a CNV signature of NSCLC. Using only their CNV values, the classification model separated the LUADs from the LUSCs with an accuracy of 0.88 and 0.84, respectively, in the training and validation datasets. The same signature also classified NSCLC tumors from their corresponding non-malignant samples with an accuracy of 0.96 and 0.98, respectively. We also compared the CNV patterns of NSCLC tumors with those of histologically similar tumors arising at other sites, such as the breast, head, and neck, and four additional tumors. Of greater importance, the significant differences between these tumors may offer the possibility of identifying the origin of tumors whose origin is unknown.

Analysis of Fifty Hotspot Mutations of Lung Squamous Cell Carcinoma in Never-smokers.

Smoking is the major risk factor for lung squamous cell carcinoma (SCC), although a small number of lung SCCs occurs in never-smokers. The purpose of this study was to compare 50 hotspot mutations of lung SCCs between never-smokers and smokers. We retrospectively reviewed the medical records of patients newly diagnosed with lung SCC between January 1, 2011 and December 31, 2013 in the Seoul National University Hospital. Formalin-fixed, paraffin-embedded tumor samples were used for analysis of hotspot mutations. Fifty cancer-related genes in never-smokers were compared to those in ever-smokers. Of 379 lung SCC patients, 19 (5.0%) were never-smokers. The median age of these 19 patients was 67 years (interquartile range 57–73 years), and 10 of these patients were women (52.5%). The incidence rates of stage I, II, III, and IV disease in this group were 26.4%, 5.3%, 31.6%, and 36.8%, respectively, and sequencing was performed successfully in 14 cases. In the 26 lung SCC tumor samples (12 from never-smokers and 14 from ever-smokers) sequenced using personal genome machine, the most common mutations were in TP53 (75.0%), RAS (66.7%), and STK11 (33.3%), but mutations were also found in EGFR, KIT, and PTEN. The distribution of hotspot mutations in never-smokers was similar to that in ever-smokers. There was no significant difference in overall survival between the 2 groups. The 50 hotspot mutations of lung SCC in never-smokers were similar to those of ever-smokers.

P2.03b-070 Establishment of Organoid Cell Lines from Lung Squamous Cell Carcinoma: Topic: Biomarkers

The limited number of available lung squamous cell carcinoma (LUSC) cell lines poses significant challenge for biological, experimental therapeutic and biomarker research in LUSC. Novel approaches to establish new preclinical models are urgently needed. We have previously established patient-derived xenografts (PDX) from resected tumors of LUSC patients and characterized them on the genomic, transcriptomic, and proteomic levels. We have used these PDX models to develop a method for establishment of 3D organoid cultures and cell lines as new in vitro preclinical models of LUSC. PDX models were established and propagated from resected primary non-small cell lung cancer (NSCLC) in NOD/SCID mice; they were molecularly profiled by exome sequencing, SNP array for copy number analysis, and immunohistochemistry (IHC). PDX tissue harvested from mice was dissociated into single cells and plated in 100% matrigel dome, with overlaying media on top. Organoids were characterized by H&E, and IHC of p63, CK5/6, TTF-1, and CK7. Organoid growth rate and drug screening were assessed using Celltiter glo cell viability assay. A total of 17 LUSC PDX models have been used for this study. All organoids were able to initiate in culture at passage 1, and the organoid establishment rate (beyond passage 4) is 50% (6/12). 4/12 (33%) LUSC organoids were able to be propagated beyond 10 passages for over 60 days with an average doubling rate of 2-3 days. Organoid tumor cells recapitulated the histological features of LUSC and were positive for p63 and CK5/6, and negative for TTF-1 and CK7 by IHC. Molecular characterization of LUSC PDX models revealed PIK3CA mutations, amplifications, and PTEN loss. Over 40% (4/9) of PI3K altered LUSC organoids were sensitive to PI3K inhibitor BKM120. LUSC organoids can be established for long term culture and recapitulate the phenotypic features of the PDX. The culture protocol is currently being tested on primary patient LUSC tumors. Organoid cultures and cell lines may be useful as additional preclinical models for functional validation of novel therapeutic targets in LUSC.

Abstract 971: A novel long noncoding RNA, onco-lncRNA 230, induces apoptosis and invasion in lung squamous cell carcinoma

Abstract Long non-coding RNAs (lncRNAs) are RNAs that are longer than 200 base pairs and do not translate into proteins but have been shown to play roles in all aspects of biological regulation. LncRNAs have also been shown to regulate gene expression including chromatin organization, transcriptional regulation, and post-transcriptional control. In addition, they are now emerging to be important players in our understanding of cancer and have been shown to promote tumorigenesis. However, the mechanisms driving the advancement of tumor progression to metastasis is poorly understood. To identify lncRNAs critical to tumorigenesis, our lab conducted a pan-cancer analysis of normal and primary tumor tissues from The Cancer Genome Atlas to discover lncRNAs that are commonly altered across cancer types, which we termed ‘onco-lncRNAs’. During our analysis, we discovered a novel unannotated onco-lncRNA, onco-lncRNA-230, that was significantly up regulated in head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC) tumors relative to matched adjacent normal tissues, when available. We chose to focus on onco-lncRNA-230 as it displayed more than seven fold increase in LUAD and more than thirty seven fold change in LUSC tumors relative to adjacent normal. First, we validated this finding by quantitative PCR which revealed greater than a two fold enrichment of onco-lncRNA-230 expression in two out of seven LUAD and all six LUSC cells lines compared to a normal lung cell line. Because we observed the greatest increase in expression in HCC95 LUSC cells, we next sought to determine if modulating the expression of onco-lncRNA-230 promotes oncogenic phenotypes. Silencing of onco-lncRNA-230 in HCC95 cells did not change cellular proliferation but significantly increased apoptosis. Furthermore, silencing onco-lncRNA-230 resulted in a decrease in cellular invasion by transwell assay. Taken together, this represents the first study reporting that onco-lncRNA-230 is dysregulated in multiple cancer types, significantly up-regulated in lung cancer patient samples and lung cancer cell lines, and confers oncogenic phenotypes. Moving forward, we intend to explore how onco-lncRNA-230 mechanistically promotes lung cancer with the objective of utilizing onco-lncRNA-230 as a novel cancer diagnostic and therapy. Citation Format: Cynthia Y. Tang, Jessica M. Silva-Fisher, Ha X. Dang, Nicole M. White, Christopher A. Maher. A novel long noncoding RNA, onco-lncRNA 230, induces apoptosis and invasion in lung squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 971.

Abstract 2264: Tumor fragmentation is a poor prognostic histologic biomarker for lung squamous cell carcinoma

Abstract Introduction: Lung squamous cell carcinoma (SCC) is the second most frequent histologic type of non-small cell lung carcinoma (NSCLC). Next to pTNM staging, grading is also important for histomorphologic classification of tumors into relevant prognostic groups. SCC forms cohesive tumor sheets of various sizes, ranging from large pushing boundaries to small detached clusters. The presence of small clusters such as single cells, tumor buds 15 cells) could also contain prognostic information and hence be pertinent for tumor grading. Herein we propose a computer-based morphometric analysis for the global assessment of lung SCC fragmentation. Methods: Surgically resected lung SCC tumors (discovery tissue microarray cohort n = 208, 2 cores per patient; validation whole section cohort n = 99, 2 blocks per patient) were immuno-histochemically stained with pan-cytokeratin. Color-based segmentation (tumor epithelia brown, stroma blue-grey) allowed to separate tumor from stroma and to compute the number of tumor fragments. Associations with disease-free (DFS) and overall survival (OS) were assessed by univariate Cox regressions. Results: Tumor fragments were defined as tumor clusters &amp;gt;3000μm2 (circa &amp;gt;15 cells) entirely surrounded by stroma. Criteria for high fragmentation were: &amp;gt; 5 fragments summed over 2 TMA cores (range: 2-18, median = 5) and &amp;gt; 700 fragments summed over 2 whole sections (range: 94-3988, median = 570). Respectively, 44% and 42% of the patients had highly fragmenting tumors in the discovery (D) and validation cohort (V), correlating with blood vessel infiltration (cc = 0.181, p&amp;lt;0.001 (D); cc = 0.220, p = 0.029 (V)). High fragmentation was also associated with decreased OS (HR = 1.77, p = 0.001) and DFS (HR = 1.83, p = 0.027) in (D). This was confirmed in (V): OS (HR = 2.09, p = 0.004), DFS (HR = 2.60, p = 0.005). Conclusions: We used an automated morphometric approach for unbiased scoring of tumor fragmentation on digitalized histological sections. This versatile method could potentially be applied to virtually any solid human carcinoma. Our analysis revealed that tumor fragmentation is a poor prognosticator for lung squamous cell carcinoma, e.g. due to increased likelihood of vessel infiltration. Citation Format: Ruben Casanova, Alex Soltermann. Tumor fragmentation is a poor prognostic histologic biomarker for lung squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2264.